Ten microliters of ligation mixture were used to transform the E. coli DH5α ( Ausubel et al., 2000). Six clones were cultured, and the plasmids were then purified using Zyppy Plasmid Miniprep (ZymoResearch). Clones were sequenced using the Big Dye Terminator V3.1 Cycle Sequence kit and fractionated on

an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). The sequencing was performed at the Biotechnology Center in the Butantan Institute, using the primers M13 (5′-GTAAAACGACGGCCAGT-3′) and T7 (5′-TAATACGACTCACTATAGGG -3′) to sequence the insert’s boundaries, and intron-def-FWD (5′-GATTATTTCTTCCCTCCTACG-3′) and intron-def-REV (5′-GACTTCCGATTCCCTGTTGC-3′) to sequence intron 1. The sequences were analyzed for selective pressure using the Hyphy package in the Datamonkey server at www.datamonkey.org CT99021 clinical trial (Pond et al., 2005). Datamonkey implements likelihood-based approaches for detecting sites under selection (Pond and Frost, 2005). Our data were analyzed using

selleckchem the following options: codon, universal code, SLAC (single likelihood ancestor counting) and REV model (time reversible model nucleotide substitution model to estimate the branch lengths and nucleotide substitution biases). Sequences were aligned in MAFFT v7.017b (Katoh and Toh, 2010), strategy E–INS–i to less than 200 sequences, with multiple conserved domains and long gaps. Gene phylogenies were constructed by maximum parsimony using TNT1.1 (Goloboff et al., 2008), by maximum likelihood using TreeFinder 1.4 (Jobb et al., 2004), and by Bayesian analysis using

MrBayes 3.2 (Ronquist et al., 2011). We Miconazole used five partitions for the probabilistic analyses (three exons and two introns), assuming the best substitution model according to AICc using TreeFinder. The reconciliation of gene tree with species tree was done in Mesquite v2.75 (Maddison and Maddison, 2011). We detected 13 β-defensin-like sequences from 12 species of Brazilian Crotalinae snakes, which are listed along with GenBank accession number in Table 1, and aligned sequences are shown in Supplementary Material 1. Despite the similarity of the nucleotide sequences, mutations in B.alternatus_sequence_01 and B.insularis_sequence_02 caused the loss of Cys which resulted in the loss of β-defensin structure and a change or loss of function. Although the sequence B.atrox_defensinB_01 showed a premature stop codon, this occurred after the sixth Cys, which did not compromise the β-defensin scaffold. B.atrox_defensinB_01 may maintain its antimicrobial function with a short C-terminal. The gene sizes varied from 852 to 2397 bp, and they were organized in three exons and two introns ( Table 2), except the DefbBa01 sequence which had only two exons. Interestingly, Oguiura et al. (2009) also described two sequences of crotamine genes without intron 2 in two rattlesnakes, indicating the possible occurrence of a minor gene structure with two exons and one intron.